Factor VIII Fusion Protein

ABSTRACT

The present invention relates to modified coagulation factors. In particular, the present invention relates to conjugated Factor VIII molecules fused to a polypeptide such as e.g. an antibody binding protein or a Fc domain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 13/574,686, filed Oct. 5, 2012, which is a 35 U.S.C. §371 National Stage application of International Application PCT/EP2011/051959 (WO 2011/101284), filed Feb. 10, 2011, which claims priority to European Patent Application 10153715.7, filed Feb. 16, 2010; this application claims priority under 35 U.S.C. §119 to U.S. Provisional Application 61/306,177; filed Feb. 19, 2010; the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to modified coagulation factors. In particular, the present invention relates to Factor VIII molecules fused to a non-homologous polypeptide such as e.g. an antibody binding polypeptide, such as e.g. an Fc receptor or an Fc domain. The invention furthermore relates to use of such molecules as well as methods for producing such molecules.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 19, 2012 and modified Sep. 30, 2015, is named 7920US01_SeqList_ST25.txt and is 90,020 bytes in size.

BACKGROUND OF THE INVENTION

Haemophilia A is an inherited bleeding disorder caused by deficiency or dysfunction of coagulation factor VIII (FVIII) activity. The clinical manifestation is not on primary haemostasis—formation of the blood clot occurs normally—but the clot is unstable due to a lack of secondary thrombin formation. The disease is treated by intravenously injection of coagulation factor FVIII which is either isolated from blood or produced recombinantly.

Current treatment recommendations are moving from traditional on-demand treatment towards prophylaxis. The circulatory half life of endogenous FVIII bound to von Willebrandt Factor is 12-14 hours and prophylactic treatment is thus to be performed several times a week in order to obtain a virtually symptom-free life for the patients. IV administration is for many, especially children and young persons, associated with significant inconvenience and/or pain.

Various methods have been employed in the development of a Factor VIII variant with significantly prolonged circulatory half life. A number of these methods relate to conjugation of Factor VIII with hydrohphilic polymers such as e.g. PEG (poly ethylene glycol).

There is thus a need in the art for novel Factor VIII products with factor VIII activity that comprise one or more of the following features: preferably homogenous in structure, preferably safe, preferably biologically degradable, and preferably have a significantly prolonged circulatory half life in order to reduce the number of factor VIII administration per week. There is likewise a need in the art for relatively simple methods for providing such molecules.

SUMMARY OF THE INVENTION

The present invention relates to a recombinant Factor VIII molecule, wherein said Factor VIII molecule is a fusion protein. The invention furthermore relates to methods for making such molecules as well as use of such molecules. Such molecules preferably have a modified circulatory half life.

DESCRIPTION OF THE INVENTION Definitions

Fusion protein: Fusion proteins/chimeric proteins, are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins. Factor VIII molecules according to the present invention may be fused to another polypeptide. Preferably, the Factor VIII fusion protein will have a longer circulatory half life compared to the non-fused Factor VIII molecule.

A wide range of fusion partners can be joined to the FVIII part. These fusion partners can alter the properties of the fusion protein relative to wild-type FVIII by various mechanisms.

A number of fusion partners are presumed to delay in vivo clearance of FVIII by interaction with the neonatal Fc receptor (FcRn). Non-limiting examples of fusion partners that assumingly protracts FVIII by interaction with FcRn are immunoglobulin Fc domains, human serum albumin (hSA), and transferrin or parts of these proteins. Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by interaction with FcRn are shown in Table 2. “Fc fusion derivatives” or “Fc fusion proteins” is herein meant to encompass FVIII variants according to the invention fused to an Fc domain that can be derived from any antibody isotype, although an IgG Fc domain will often be preferred due to the relatively long circulatory half life of IgG antibodies. The Fc domain may furthermore be modified in order to modulate certain effector functions such as e.g. complement binding and/or binding to certain Fc receptors. Fusion of a FVIII with an Fc domain, having the capacity to bind to FcRn receptors, will generally result in a prolonged circulatory half life of the fusion protein compared to the half life of the wt FVIII. Mutations in positions 234, 235 and 237 in an IgG Fc domain will generally result in reduced binding to the FcγRI receptor and possibly also the FcγRIIa and the FcγRIII receptors. These mutations do not alter binding to the FcRn receptor, which promotes a long circulatory half life by an endocytic recycling pathway. Preferably, a modified IgG Fc domain of a fusion protein according to the invention comprises one or more of the following mutations that will result in decreased affinity to certain Fc receptors (L234A, L235E, and G237A) and in reduced C1q-mediated complement fixation (A330S and P331S), respectively.

However, the present invention also includes FVIII variants fused to Fc domains having altered binding properties to e.g. the neonatal Fc receptors. If e.g. Fc domain variants have increased affinity to e.g. the neonatal Fc receptor, it is plausible that the in vivo circulatory half life of the fusion protein will be further improved. Examples of amino acid replacements in human IgG believed to modulate the affinity of the Fc domain to the neonatal Fc receptor are T250Q, M252Y, S254T, T256E, P257I, T307A, Q311I, and M428L (residue numbering according to the EU index).

Other fusion partners are presumed to delay in vivo clearance of FVIII by interaction with immunoglobulins. A non-limiting example of fusion partners that assumingly protracts FVIII by interaction with immunoglobulins is Fc receptors such as Fc␣RI (CD64) or FcRn or parts of these proteins. Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by interaction with immunoglobulins are shown in Table 3.

Some fusion partners are presumed to delay in vivo clearance of FVIII by reducing the interaction with clearance receptors. A non-limiting example of fusion partners that assumingly protracts FVIII by reducing the interaction with clearance receptors is members of the low-density lipoprotein receptor family Fc receptors such as low-density lipoprotein receptor-related protein or parts of these proteins for instance for instance cluster of repeat 5 (CR5), 6 (CR6), and/or 7 (CR7). Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by reducing the interaction with clearance receptors are shown in Table 4.

Other fusion partners are presumed to delay in vivo clearance of FVIII by interaction with platelets. A non-limiting example of fusion partners that assumingly protracts FVIII by interaction with platelets is single-chain (SC) antibodies binding to proteins on the platelet surface such as GPIIIa. In SC antibodies, the polypeptide sequence derived from immunoglobulin heavy chain can be situated N-terminal to the polypeptide sequence derived from immunoglobulin light chain. This order is referred to as HC-LC. The polypeptide sequence derived from immunoglobulin heavy chain can also be situated C-terminal to the polypeptide sequence derived from immunoglobulin light chain. The latter order is referred to as LC-HC. Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by interaction with platelets are shown in Table 5.

Some fusion partners are presumed to delay in vivo clearance of FVIII by interaction with serum albumin. Non-limiting examples of fusion partners that assumingly protracts FVIII by interaction with serum albumin are single-chain anti-serum albumin antibodies (SC anti-HSA) and albumin-binding polypeptides such as the ABD035 polypeptide. The albumin-binding polypeptides can be repeated several times, for instance 4 repetitions as present in 4XABD035 fusion partner. Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by interaction with serum albumin are shown in Table 6.

Other fusion partners are presumed to delay in vivo clearance of FVIII by shielding. A non-limiting example of fusion partners that assumingly protracts FVIII by shielding is polypeptides with stretches of non-hydrophobic amino acids such as Sequence A (seq A) or repletion of elastin-like polypeptide (ELP) for instance ELP60. Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by shielding are shown in Table 7.

Some fusion partners are presumed to delay in vivo clearance of FVIII by modulating the affinity to vWF. A non-limiting example of fusion partners that assumingly protracts FVIII by modulating the affinity to vWF is the a3 region of FVIII (amino acid 1649-1689 of wild-type human FVIII) or parts of the a3 region, thus adding one or more extra a3 regions to FVIII. Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by modulating the affinity to vWF are shown in Table 8.

Some fusion partners are presumed to delay in vivo clearance of FVIII by mechanisms remaining to be determined Non-limiting examples of fusion partners that assumingly protracts FVIII by mechanisms remaining to be determined are growth hormone binding protein (GHBP), parts of coagulation factor IX (FIX), parts of vWF, vWF binding protein, parts of chorion gonadotropin, and parts of coagulation factor X (FX). Non limiting examples of fusion partners derived from FIX are amino acid 298-342 of human FIX (FIX298-342) and amino acid 47-125 of human FIX (FIX47-125). Non-limiting examples of fusion partners derived from vWF are amino acid 1-272 of human vWF (vWF1-272), amino acid 1-1390 of human vWF (vWF1-1390, and amino acid 497-716 of human vWF (vWF497-716). A non-limiting example of fusion partners derived from chorion gonadotropin is the C-terminal 28 amino acids of the beta-chain of human chorion gonadotropin (hCG C-terminus). A non-limiting example of fusion partners derived from FX is the activation peptide of human FX (F10AP). Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by mechanisms remaining to be determined are shown in Table 9.

Some fusion partners are presumed to delay in vivo clearance of FVIII by modulating the affinity to lipids. A non-limiting example of fusion partners that assumingly protracts FVIII by modulating the affinity to lipids is the C2 domain of FVIII, thus adding one or more extra C2 domains to FVIII. Non-limiting examples of fusion proteins consisting of a FVIII part joined to polypeptides assumed to protract FVIII by modulating the affinity to lipids are shown in Table 10

Fc receptor: Fc receptors are cell surface receptors that recognize and bind the Fc portion of antibodies. Based on structure, cell distribution and affinity to IgG, the Fc receptors are divided into three classes: FcγRI (CD64), FcγRII (CD32), and FCγRIII (CD16). According to the present invention, the Fc receptors fused to Factor VIII molecules may be of full length or partial length, as well as any variants thereof (variants include amino acid substitutions, deletions and additions). If they are of partial length it follows that the ability to bind antibodies should be retained.

Von Willebrandt Factor (vWF): vWF is a large mono-/multimeric glycoprotein present in blood plasma and produced constitutively in endothelium (in the Weibel-Palade bodies), megakaryocytes (α-granules of platelets), and subendothelial connective tissue. Its primary function is binding to other proteins, particularly Factor VIII and it is important in platelet adhesion to wound sites.

Factor VIII is bound to vWF while inactive in circulation; Factor VIII degrades rapidly or is cleared when not bound to vWF. It thus follows that reduction or abolishment of vWF binding capacity in FVIII has thus far been considered as a highly undesirable approach in obtaining Factor FVIII variants with prolonged circulatory half life.

The term “reduced capacity to bind vWF” is herein meant to encompass Factor VIII variants, wherein the capacity to bind vWF is decreased by at least 50%, preferably by at least 60%, more preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, and most preferably about 100%. FVIII binding to vWF may be measured either by an ELISA like assay or as direct binding to immobilized vWF using surface plasmon resonance. The region in Factor VIII responsible for binding to vWF is the region spanning residues 1670-1684 as disclosed in EP0319315. It is envisaged that Factor VIII point and/or deltion mutatants involving this area will modify the ability to bind to vWF. Examples of particularly preferred point mutations according to the present invention include variants comprising one or more of the following point mutations: Y1680F, Y1680R, Y1680N, and E1682T, and Y1680C.

Factor VIII molecules: FVIII/Factor VIII is a large, complex glycoprotein that primarily is produced by hepatocytes. FVIII consists of 2351 amino acids, including signal peptide, and contains several distinct domains, as defined by homology. There are three A-domains, a unique B-domain, and two C-domains. The domain order can be listed as NH2-A1-A2-B-A3-C1-C2-COOH. FVIII circulates in plasma as two chains, separated at the B-A3 border. The chains are connected by bivalent metal ion-bindings. The A1-A2-B chain is termed the heavy chain (HC) while the A3-C1-C2 is termed the light chain (LC).

Endogenous Factor VIII molecules circulate in vivo as a pool of molecules with B domains of various sizes. What probably occurs in vivo is a gradual enzymatic removal of the B domain resulting in a pool of molecules with B-domains of various sizes. It is generally believed that cleavage at position 740, by which the last part of the B-domain is removed, occurs in connection with thrombin activation. However, it cannot be ruled out that a Factor VIII variant in which e.g. the cleavage site at position 740 has been impaired may be active.

“Factor VIII” or “FVIII” as used herein refers to a human plasma glycoprotein that is a member of the intrinsic coagulation pathway and is essential to blood coagulation. “Native FVIII” is the full length human FVIII molecule as shown in SEQ ID NO. 1 (amino acid 1-2332). The B-domain is spanning amino acids 741-1648 in SEQ ID NO 1.

SEQ ID NO 1: ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYK KTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASH PVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVL KENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEK TQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVN GYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHR QASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSC PEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAK KHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQAS RPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTV EDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGN QIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQAS NIMHSINGYFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALL KVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQ KQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTP HGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDM VFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNL AAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEE NNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDN ALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILES DTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKE GPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQL VSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFL TNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNF MKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSK KGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQ FRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGA ITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQD NSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQRE VGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDL FPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATE SSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTIL SLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRH QREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQK KTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTD GSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYS SLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKA WAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFT IFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDT LPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMAL YNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQ TPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS WIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYR GNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLR MELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYV KEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTR YLRIHPQSWVHQIALRMEVLGCEAQDLY

The factor VIII molecules according to the present invention may be B domain truncated Factor FVIII molecules wherein the remaining domains correspond closely to the sequence as set forth in amino acid no 1-740 and 1649-2332 in SEQ ID NO 1 although there may also e.g. be one or more alterations within the vWF binding region between residues 1670-1684. However, B domain truncated molecules according to the invention may differ slight from the sequence set forth in SEQ ID NO 1, meaning that the remaining domains (i.e. the three A-domains and the two C-domains) may differ slightly e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or about 1%, 2%, 3%, 4% or 5% from the amino acid sequence as set forth in SEQ ID NO 1 (amino acids 1-740 and 1649-2332) due to the fact that mutations can be introduced in order to reduce e.g. vWF binding capacity. Furthermore, it is plausible that amino acid modifications (substitutions, deletions, etc.) are introduced other places in the molecule in order to modify the binding capacity of Factor VIII with various other components such as e.g. LRP, various receptors, other coagulation factors, cell surfaces, introduction and/or abolishment of glycosylation sites, etc.

Factor VIII molecules according to the present invention have Factor VIII activity, meaning the ability to function in the coagulation cascade in a manner functionally similar or equivalent to FVIII, induce the formation of FXa via interaction with FIXa on an activated platelet, and support the formation of a blood clot. The activity can be assessed in vitro by techniques well known in the art such as e.g. clot analysis, endogenous thrombin potential analysis, etc. Factor VIII molecules according to the present invention have FVIII activity being at least about 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and 100% or even more than 100% of that of native human FVIII.

B domain: The B-domain in Factor VIII spans amino acids 741-1648 in SEQ ID NO 1. The B-domain is cleaved at several different sites, generating large heterogeneity in circulating plasma FVIII molecules. The exact function of the heavily glycosylated B-domain is unknown. What is known is that the domain is dispensable for FVIII activity in the coagulation cascade. This apparent lack of function is supported by the fact that B domain deleted/truncated FVIII appears to have in vivo properties identical to those seen for full length native FVIII. That being said there are indications that the B-domain may reduce the association with the cell membrane, at least under serum free conditions.

B domain truncated/deleted Factor VIII molecule: Endogenous full length FVIII is synthesized as a single-chain precursor molecule. Prior to secretion, the precursor is cleaved into the heavy chain and the light chain. Recombinant B domain-deleted FVIII can be produced from two different strategies. Either the heavy chain without the B-domain and the light chain are synthesized individually as two different polypeptide chains (two-chain strategy) or the B-domain deleted FVIII is synthesized as a single precursor polypeptide chain (single-chain strategy) that is cleaved into the heavy and light chains in the same way as the full-length FVIII precursor.

In a B domain-deleted FVIII precursor polypeptide, the heavy and light chain moieties are normally separated by a linker To minimize the risk of introducing immunogenic epitopes in the B domain-deleted FVIII, the sequence of the linker is preferable derived from the FVIII B-domain. As a minimum, the linker must comprise a recognition site for the protease that separates the B domain-deleted FVIII precursor polypeptide into the heavy and light chain. In the B domain of full length FVIII, amino acid 1644-1648 constitutes this recognition site. The thrombin site leading to removal of the linker on activation of B domain-deleted FVIII is located in the heavy chain. Thus, the size and amino acid sequence of the linker is unlikely to influence its removal from the remaining FVIII molecule by thrombin activation. Deletion of the B domain is an advantage for production of FVIII. Nevertheless, parts of the B domain can be included in the linker without reducing the productivity. The negative effect of the B domain on productivity has not been attributed to any specific size or sequence of the B domain.

The truncated B-domain may contain several O-glycosylation sites. However, according to a preferred embodiment, the molecule comprises only one, alternatively two, three or four O-linked oligosaccharides in the truncated B-domain.

According to a preferred embodiment, the truncated B domain comprises only one potential O-glycosylation sites and a side chain, such as e.g. PEG or an albumin binder is covalently conjugated to this O-glycosylation site.

The O-linked oligosaccharides in the B-domain truncated molecules according to the invention may be attached to O-glycosylation sites that were either artificially created by recombinant means and/or by exposure of “hidden” O-glycosylation sites by truncation of the B-domain. In both cases, such molecules may be made by designing a B-domain truncated Factor VIII amino acid sequence and subsequently subjecting the amino acid sequence to an in silico analysis predicting the probability of O-glycosylation sites in the truncated B-domain. Molecules with a relatively high probability of having such glycosylation sites can be synthesized in a suitable host cell followed by analysis of the glycosylation pattern and subsequent selection of molecules having O-linked glycosylation in the truncated B-domain.

The Factor VIII molecule also contains a number of N-linked oligosaccharides and each of these may likewise serve as an anchor for attachment of a hydrophobic side group.

Suitable host cells for producing recombinant factor VIII fusion proteins according to the invention are preferably of mammalian origin in order to ensure that the molecule is glycosylated. In practicing the present invention, the cells are mammalian cells, more preferably an established mammalian cell line, including, without limitation, CHO, COS-1, baby hamster kidney (BHK), and HEK293 cell lines. Other suitable cell lines include, without limitation, Rat Hep I, Rat Hep II, TCMK, Human lung, DUKX cells (CHO cell line) Also useful are 3T3 cells, Namalwa cells, myelomas and fusions of myelomas with other cells. In some embodiments, the cells may be mutant or recombinant cells, such as, e.g., cells that express a qualitatively or quantitatively different spectrum of enzymes that catalyze post-translational modification of proteins (e.g., glycosylation enzymes such as glycosyl transferases and/or glycosidases, or processing enzymes such as propeptides) than the cell type from which they were derived. DUKX cells (CHO cell line) are especially preferred.

Currently preferred cells are HEK293, COS, Chinese Hamster Ovary (CHO) cells, Baby Hamster Kidney (BHK) and myeloma cells, in particular Chinese Hamster Ovary (CHO) cells.

The length of the B domain in the wt FVIII molecule is about 908 amino acids. The length of the truncated B domain in molecules according to the present invention may vary from about 10 to about 800 amino acids, such as e.g. from about 10 amino acids to about 700 acids, such as e.g. about 12-500 amino acids, 12-400 amino acids, 12-300 amino acids, 12-200 amino acids, 15-100 amino acids, 15-75 amino acids, 15-50 amino acids, 15-45 amino acids, 20-45 amino acids, 20-40 amino acids, or 20-30 amino acids. The truncated B-domain may comprise fragments of the heavy chain and/or the light chain and/or an artificially introduced sequence that is not found in the wt FVIII molecule. The terms “B-domain truncated” and “B-domain deleted” may be used interchangeably herein.

Modified circulatory half life: Molecules according to the present invention have a modified circulatory half life compared to the wild type Factor VIII molecule, preferably an increased circulatory half life. Circulatory half life is preferably increased at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 100%, more preferably at least 125%, more preferably at least 150%, more preferably at least 175%, more preferably at least 200%, and most preferably at least 250% or 300%. Even more preferably, such molecules have a circulatory half life that is increased at least 400%, 500%, 600%, or even 700%.

Hydrophilic polymer: The side group according to the present invention is preferably a non-naturally occurring hydrophilic polymer comprising at least one non-naturally occurring polymeric moiety. In another example, the non-naturally occurring modifying group is a modified carbohydrate.

Exemplary hydrophilic polymers according to the invention include water soluble polymers that can be linear or branched and can include one or more independently selected polymeric moieties, such as poly(alkylene glycol) and derivatives thereof. The polymeric modifying group according to the invention may include a water-soluble polymer, e.g. poly(ethylene glycol) and derivatived thereof (PEG, m-PEG), poly(propylene glycol) and derivatives thereof (PPG, m-PPG) and the like.

The polymer backbone of the water-soluble polymer according to the invention can be poly(ethylene glycol) (i.e. PEG). The term PEG in connection with the present invention includes poly(ethylene glycol) in any of its forms, including alkoxy PEG, difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG (i.e. PEG or related polymers having one or more functional groups pendent to the polymer backbone), or PEG with degradable linkages therein.

The polymer backbone can be linear or branched. Branched polymer backbones are generally known in the art. Typically, a branched polymer has a central branch core moiety and a plurality of linear polymer chains linked to the central branch core. PEG is commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, pentaerythritol and sorbitol. The central branch moiety can also be derived from several amino acids, such as lysine or cysteine. In one example, the branched poly(ethylene glycol) can be represented in general form as R(-PEG-OH)m in which R represents the core moiety, such as glycerol or pentaerythritol, and m represents the number of arms. Multi-armed PEG molecules, such as those described in U.S. Pat. No. 5,932,462, which is incorporated by reference herein in its entirety, can also be used as the polymer backbone.

Many other polymers are also suitable for the invention. Polymer backbones that are non-peptidic and water-soluble, are particularly useful in the invention. Examples of suitable polymers include, but are not limited to, other poly(alkylene glycols), such as poly(propylene glycol) (“PPG”), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefmic alcohol), poly(vinylpyrrolidone), poly(hydroxypropylmethacrylamide), poly([alpha]-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), such as described in U.S. Pat. No. 5,629,384, which is incorporated by reference herein in its entirety, as well as copolymers, terpolymers, and mixtures thereof.

Although the molecular weight of each chain of the polymer backbone can vary, it is typically in the range of from about 100 Da to about 160,000 Da, such as e.g. from about 5,000 Da to about 100,000 Da. More specifically, the size of each conjugated hydrophilic polymer according to the present invention may vary from about 500 Da to about 80,000 Da, such as e.g. about 1000 Da to about 80,000 Da; about 2000 Da to about 70,000 Da; about 5000 to about 70,000 Da; about 5000 to about 60,000 Da; about 10,000 to about 70,000 Da; about 20,000 to about 60,000 Da; about 30,000 to about 60,000 Da; about 30,000 to about 50,000 Da; or about 30,000 to about 40,000 Da. It should be understood that these sizes represent estimates rather than exact measures. According to a preferred embodiment, the molecules according to the invention are conjugated with a heterogenous population of hydrophilic polymers, such as e.g. PEG of a size of e.g. 10,000, 40,000, or 80,000 Da+/−about 5000, about 4000, about 3000, about 2000, or about 1000 Da.

Side chain/side group: The Factor VIII molecules according to the present invention may also be conjugated with side groups other than hydrophilic polymers. The side chain according to the present invention may e.g. be selected from one or more of the list consisting of: fatty acids and derivates thereof (sometimes referred to as “albumin binders”), peptides, etc., The hydrophobic side groups according to the invention are preferably biologically degradable. There may furthermore be more than one hydrophobic side group conjugated to an individual Factor VIII molecule, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. It furthermore follows that it is possible to conjugate Factor VIII molecules according to the invention with one or more hydrophobic side groups as well as one or more hydrophobic side groups, peptidic side groups, etc.

An individual Factor VIII molecule according to the invention may thus comprise side groups of e.g. both hydrophilic and hydrophobic/peptidic nature. It is nevertheless associated with additional efforts to conjugate the molecule with different types of side groups and such solutions may thus turn out to be relatively impractical in practice. The molecules according to the present invention thus preferably comprise only one type of side chains (e.g. hydrophilic/hydrophobic side chains).

Conjugation of Factor VIII with hydrophobic side groups has thus far, however, not been considered being an attractive alternative to conjugation with hydrophilic groups. One explanation may be that laborious and/or harsh chemical methods employing organic solvents would have been expected to be required. Another explanation may be that the usually relatively small hydrophobic molecules cannot be expected to be efficient in binding a conjugated (large) Factor VIII molecule to e.g. albumin and thereby possibly shielding the molecule from clearance.

Albumin Binder Conjugates

It is known that the in vivo properties of such proteins can be improved by the use of albumin binding side chains. Such side chains, or albumin binders, can be attached to the protein prior to administration and can, for example, stabilise the protein in vivo or improve or extend the in vivo half-life of the protein.

The albumin binder may thereby promote the circulation of the derivative with the blood stream. The albumin binder may have the effect of extending or protracting the time of action of the protein that it is bound to it, due to the fact that the complexes of the peptide derivative and albumin are only slowly disintegrated to release the active pharmaceutical ingredient. Thus, a preferred substituent, or side chain, as a whole may be referred to as an albumin binding moiety.

The albumin binder (albumin binding moiety) may comprise a portion which is particularly relevant for the albumin binding and thereby the protraction of circulation in the blood stream, which portion may accordingly be referred to as a protracting moiety. The protracting moiety is preferably at, or near, the opposite end of the albumin binding moiety as compared to its point of attachment to the peptide.

In a preferred embodiment, the albumin binder is, or comprises, a side chain that is capable of forming non-covalent complexes with albumin. The albumin binder may bind albumin non-covalently and/or reversibly. The albumin binder may bind albumin specifically. As is clear from the methods described below, the albumin binder may bind to cyclodextrin. The albumin binder may bind cyclodextrin non-covalently and/or reversibly. The albumin binder may bind cyclodextrin specifically.

An albumin binder as described herein is generally a hydrophobic group.

The other portion of the albumin binding moiety, i.e. the portion in-between the protracting moiety and the point of attachment to the peptide, may be referred to as a linker moiety, linker, spacer, or the like. However, the presence of such a linker is optional, and hence the albumin binding moiety may be identical to the protracting moiety.

In particular embodiments, the albumin binding moiety and/or the protracting moiety is lipophilic, and/or negatively charged at physiological pH (7.4).

The albumin binding moiety and/or the protracting moiety may be covalently attached to an amino group of the peptide by conjugation chemistry such as by alkylation, acylation, or amide formation; or to a hydroxyl group, such as by esterification, alkylation, oximation.

In a preferred embodiment, an active ester of the albumin binding moiety and/or the protracting moiety is covalently linked to an amino group of a sialic acid residue or a sialic acid derivative, under formation of an amide bond (this process being referred to as acylation).

Unless otherwise stated, when reference is made to an acylation of a protein, it is understood to be to an amino-group linked to a sialic acid residue on on glycoprotein.

For the present purposes, the terms “albumin binding moiety”, “protracting moiety”, and “linker” include the un-reacted as well as the reacted forms of these molecules. Whether or not one or the other form is meant is clear from the context in which the term is used.

The albumin binding moiety may be, or may comprise a fatty acid or fatty diacid or a derivative or either thereof.

The term “fatty acid” refers to aliphatic monocarboxylic acids having from 4 to 28 carbon atoms, such as 16 carbon atoms. It is preferably unbranched, and/or even numbered, and it may be saturated or unsaturated.

The term “fatty diacid” refers to fatty acids as defined above but with an additional carboxylic acid group in the omega position. Thus, fatty diacids are dicarboxylic acids.

The nomenclature is as is usual in the art, for example —COOH, as well as HOOC—, refers to carboxy; —C₆H₄— to phenylen; —CO—, as well as —OC—, to carbonyl (O═C<); and C₆H₅—O— to phenoxy.

In a preferred embodiment the linker moiety, if present, has from 2 to 80 C-atoms, preferably from 5 to 70 C-atoms. In additional preferred embodiments, the linker moiety, if present, has from 4 to 20 hetero atoms, preferably from 2 to 40 hetero atoms, more preferably from 3 to 30 hetero atoms. Particularly preferred examples of hetero atoms are N-, and O-atoms. H-atoms are not hetero atoms.

In another embodiment, the linker comprises at least one OEG molecule, and/or at least one glutamic acid residue, or rather the corresponding radicals (OEG designates 8-amino-3,6-dioxaoctanic acid, i.e. this radical: —NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—CO—).

In one preferred embodiment, the linker moiety comprises a di-carboxyl residue linked to a sialic acid residue by an amide bond. In preferred examples, the di-carboxyl residue has from 2-30 C-atoms, preferably 4-20 C-atoms, more preferably 4-10 C-atoms. In additional preferred examples, the di-carboxyl residue has from 0-10 hetero-atoms, preferably 0-5 hetero-atoms.

In another preferred example, the linker moiety comprises a group containing both an amino and a distal carboxyl-group linked to a sialic acid residue by an amide bond through its distal carboxyl groups. In one preferred embodiment the this group is an OEG group.

The amino acid glutamic acid (Glu) comprises two carboxylic acid groups. Its gamma-carboxy group is preferably used for forming an amide bond with an amino group of a sialic acid residue or a sialic acid derivative, or with an amino group of an OEG molecule, if present, or with the amino group of another Glu residue, if present. The amino group of Glu in turn forms an amide bond with the carboxy group of the protracting moiety, or with the carboxy group of an OEG molecule, if present, or with the gamma-carboxy group of another Glu, if present. This way of inclusion of Glu is occasionally briefly referred to as “gamma-Glu”.

Without being bound by theory it is envisaged that the reason why it may be advantageous to attach hydrophobic side groups to Factor VIII molecules with reduced vWF binding capacity rather than attaching such side groups to Factor VIII molecules with normal vWF binding capacity is that the relative size of the side group is relatively small in the large Factor VIII/vWF complex. It is hypothesized that a relatively large side group functions more efficiently in shielding the free Factor VIII from clearance. It is further hypothesized that the half life of FVIII is related to that of vWF. FVIII molecules with reduced ability to bind vWF most likely have exposed clerance epitopes which would normally have been shielded by vWF. By attaching side groups it is thus hypothesized that this “clearance shielding” can be regained. In other cases, attachment of side groups such as e.g. antibody fragments may function by e.g. attaching the molecule to proteins, cells, or platelets having a relatively long circulatory half life.

O-linked oligosaccharide: Both N-glycans and O-glycans are attached to proteins by the cells producing the protein. The cellular N-glycosylation machinery recognizes and glycosylates N-glycosylation signals (N-X-S/T motifs) in the amino acid chain, as the nascent protein is translocated from the ribosome to the endoplasmic reticulum (Kiely et al. 1976; Glabe et al. 1980).

Likewise, O-glycans are attached to specific O-glycosylation sites in the amino acid chain, but the motifs triggering O-glycosylation are much more heterogenous than the N-glycosylation signals, and our ability to predict O-glycosylation sites in amino acid sequences is still inadequate (Julenius et al. 2004). The construction of artificial O-glycosylation sites it is thus associated with some uncertainty.

Linkage of hydrophobic side groups: Conjugation of Factor FVIII molecules with hydrophobic side groups may be made using chemical methods. However, a number of advantages are potentially associated with employment of an enzymatic approach. According to a preferred enzymatic method according to the present invention, [hydrophobic protractor group]-sialyl-CMP substrate can be prepared chemically. This substrate can be transferred enzymatically to glycans present on Factor FVIII using a sialyltransferase enzyme. The inventors of the present invention have surprisingly demonstrated that this enzymatic approach can be performed without addition of any organic solvents that may. A number of disadvantages are associated with use of organic solvents, e.g. loss of biological activity, environmental concerns, additional steps to be taken to ensure that the organic solvents are completely removed, etc. It may also be possible to avoid addition of cyclodextrin—cyclodextrin is a detergent which may also contribute to loss of biological function. In the process of enzymatic conjugation, it may be an advantage to add glycerol, e.g. 5-30% glycerol, preferably 10-20% glycerol. The presence of glycerol seem to stabilize the Factor VIII molecule e.g. in the freeze/thaw process and it may also prevent formation of Factor VIII crystals. Glycerol present during enzymatic conjugation does thus not need to be removed after the conjugation process has been completed.

Sialyltransferase: Sialyltransferases are enzymes that transfer sialic acid to nascent oligosaccharide. Each sialyltransferase is specific for a particular sugar substrate. Sialyltransferases add sialic acid to the terminal portions of the sialylated glycolipids (gangliosides) or to the N- or O-linked sugar chains of glycoproteins. There are about twenty different sialyltransferases which can be distinguished on the basis of the acceptor structure on which they act and on the type of sugar linkage they form. Preferred sialyltransferases according to the present invention are ST3Gal-I (specific for O-glycans) and ST3Gal-III (specific for N-glycans). It is thus possible to engineer the structure of the conjugated Factor VIII molecules according to the present invention by e.g. selection of a specific sialyltransferase and/or engineering of a Factor VIII molecule with a particular glycosylation pattern.

Pharmaceutical composition: A pharmaceutical composition is herein preferably meant to encompass compositions comprising Factor VIIII molecules according to the present invention suitable for parenteral administration, such as e.g. ready-to-use sterile aqueous compositions or dry sterile compositions that can be reconstituted in e.g. water or an aqueous buffer. The compositions according to the invention may comprise various pharmaceutically acceptable excipients, stabilizers, etc.

Additional ingredients in such compositions may include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (e.g., human serum albumin, gelatine or proteins) and a zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine). Such additional ingredients, of course, should not adversely affect the overall stability of the pharmaceutical formulation of the present invention. Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a solution or suspension for the administration of the FVIII compound in the form of a nasal or pulmonal spray. As a still further option, the pharmaceutical compositions containing the FVIII compound of the invention may also be adapted to transdermal administration, e.g. by needle-free injection or from a patch, optionally an iontophoretic patch, or transmucosal, e.g. buccal, administration.

The term “treatment”, as used herein, refers to the medical therapy of any human or other animal subject in need thereof. Said subject is expected to have undergone physical examination by a medical practitioner, who has given a tentative or definitive diagnosis which would indicate that the use of said specific treatment is beneficial to the health of said human or other animal subject. The timing and purpose of said treatment may vary from one individual to another, according to the status quo of the subject's health. Thus, said treatment may be prophylactic, palliative, symptomatic and/or curative.

In a first aspect, the present invention relates to a recombinant Factor VIII molecule, wherein said Factor VIII molecule is a fusion protein comprising a Factor VIII molecule and a fusion partner. In a first embodiment, the fusion partner is replacing the a3-domain of the Factor VIII molecule. The small a3-domain of Factor VIII may thus be fully or partly replaced by the fusion partner. In a second embodiment, the fusion partner is inserted into the B-domain of Factor VIII. The B-domain may be a full length B-domain but in a preferred embodiment, the B domain is significantly truncated. There will thus be at least three, four, five, six, seven, eight, nine or ten amino acids originating from the B-domain at both the N- and the C-terminal ends of the fusion partner. In another preferred embodiment, the fusion partner is inserted in or at the C-terminal end of the C2 domain in Factor FVIII.

In a third embodiment, the Factor VIII molecule according to the invention is fused to an antibody binding molecule such as e.g. an Fc receptor. Examples of antibody binding molecules are listed in the tables below. In a fourth embodiment, the Factor VIII molecule is fused to a molecule having the capability of binding to human serum albumin. In another embodiment, the Factor VIII is fused to transferrin. Examples thereof are also provided below. In a fifth embodiment, the Factor VIII molecule is fused with a molecule having the capability of binding to platelets—specific examples of such molecules are likewise provided below. In yet another embodiment, the Factor VIII molecule is fused to a molecule with the capacity of binding to a Factor VIII clearance receptor.

In a sixth embodiment, the Factor VIII molecule according to the invention has reduced vWF binding capacity. Preferably, such Factor VIII molecules comprise a mutation (substitution, deletion or addition of amino acids) within the area spanning amino acids 1670-1684 in SEQ ID NO:1. Most preferably, such Factor VIII molecules comprise one of the following point mutations: Y1680F, Y1680R, Y1680N, and E1682T, and Y1680C.

In a seventh embodiment, the molecule according to the invention is conjugated with a side group. This side group may be selected from one or more of the list consisting of: hydrophilic polymers, peptides and hydrophobic side groups. Preferably, the side group is a PEG group, a fatty acid derivative, or a polypeptide. Preferably, such side groups are attached to the molecule using enzymatic approaches, such as e.g. the technology disclosed in WO0331464, wherein O-linked and/or N-linked glycans are used as linkers.

Another aspect of the present invention relates to FVIII molecule fused to a fusion partner, wherein the fusion partner is replacing the A3-domain of the Factor VIII molecule.

In one embodiment, the fusion partner is albumin. In another embodiment, the fusion partner is an Fc receptor. In another embodiment, the Fc receptor is FcγRI. In another embodiment, the fusion partner is an Fc domain. In another embodiment, the Fc domain is a mutated Fc domain having reduced effector functions and/or increased affinity to the neonatal Fc receptor. In another embodiment, the fusion protein is conjugated with a side group. In another embodiment, the side group is linked to the fusion protein via an N-linked and/or an O-linked glycan. In another embodiment, the side group is linked to an N-linked and/or an O-linked glycan via a sialic acid. In another embodiment, the side group is selected from one or more of the list consisting of hydrophilic polymers, peptides, and hydrophobic side groups. In another embodiment, the FVIII molecule is a B domain truncated molecule wherein the B domain comprises the sequence as set forth in SEQ ID NO: 2. In another embodiment, the fusion protein comprises a side group linked to the O-linked glycan in the truncated B domain that comprises the amino acid sequence as set forth in SEQ ID NO: 2. In another embodiment, the Factor VIII molecule has reduced vWF binding capacity. In another embodiment, the Factor VIII molecule having reduced vWF binding capacity comprises a mutation selected from the list consisting of: Y1680F, Y1680R, Y1680N, and E1682T, and Y1680C.

Another aspect of the present invention relates to a FVIII molecule fused to a fusion partner, wherein the fusion partner is inserted into the B-domain of Factor VIII.

In one embodiment, the fusion partner is albumin. In another embodiment, the fusion partner is an Fc receptor. In another embodiment, the Fc receptor is FcγRI. In another embodiment, the fusion partner is an Fc domain. In another embodiment, the Fc domain is a mutated Fc domain having reduced effector functions and/or increased affinity to the neonatal Fc receptor. In another embodiment, the fusion protein is conjugated with a side group. In another embodiment, the side group is linked to the fusion protein via an N-linked and/or an O-linked glycan. In another embodiment, the side group is linked to an N-linked and/or an O-linked glycan via a sialic acid. In another embodiment, the side group is selected from one or more of the list consisting of hydrophilic polymers, peptides, and hydrophobic side groups. In another embodiment, the FVIII molecule is a B domain truncated molecule wherein the B domain comprises the sequence as set forth in SEQ ID NO: 2. In another embodiment, the fusion protein comprises a side group linked to the O-linked glycan in the truncated B domain that comprises the amino acid sequence as set forth in SEQ ID NO: 2. In another embodiment, the Factor VIII molecule has reduced vWF binding capacity. In another embodiment, the Factor VIII molecule having reduced vWF binding capacity comprises a mutation selected from the list consisting of: Y1680F, Y1680R, Y1680N, and E1682T, and Y1680C.

Another aspect of the present invention relates to a FVIII molecule fused to a fusion partner, wherein the fusion partner is inserted in the C-terminal end of the C2 domain in Factor FVIII.

In one embodiment, the fusion partner is albumin. In another embodiment, the fusion partner is an Fc receptor. In another embodiment, the Fc receptor is FcγRI. In another embodiment, the fusion partner is an Fc domain. In another embodiment, the Fc domain is a mutated Fc domain having reduced effector functions and/or increased affinity to the neonatal Fc receptor. In another embodiment, the fusion protein is conjugated with a side group. In another embodiment, the side group is linked to the fusion protein via an N-linked and/or an O-linked glycan. In another embodiment, the side group is linked to an N-linked and/or an O-linked glycan via a sialic acid. In another embodiment, the side group is selected from one or more of the list consisting of hydrophilic polymers, peptides, and hydrophobic side groups. In another embodiment, the FVIII molecule is a B domain truncated molecule wherein the B domain comprises the sequence as set forth in SEQ ID NO: 2. In another embodiment, the fusion protein comprises a side group linked to the O-linked glycan in the truncated B domain that comprises the amino acid sequence as set forth in SEQ ID NO: 2. In another embodiment, the Factor VIII molecule has reduced vWF binding capacity. In another embodiment, the Factor VIII molecule having reduced vWF binding capacity comprises a mutation selected from the list consisting of: Y1680F, Y1680R, Y1680N, and E1682T, and Y1680C.

Another aspect relates to a method of making a molecule according to the present invention, wherein said method comprises incubating a host cell encoding said molecule under appropriate conditions. Accordingly, the present invention also relates to nucleic acids molecules as well as expression vectors and host cells comprising nucleic acid sequences that encode a molecule according to the present invention. Molecules obtained by or obtainable by such methods are likewise an aspect of the present invention.

Another aspect relates to use of a molecule according to the present invention as a medicine.

Another aspect relates to use of a molecule according to the present invention for treatment of haemophilia, preferably haemophilia A.

Another aspect relates to a pharmaceutical composition comprising a molecule according to the invention and optionally one or more pharmaceutically acceptable excipients.

Another aspect of the invention relates to a method of treatment of a hemophilic disease comprising administering to a patient in need thereof a therapeutically effective amount of a molecule according to the present invention.

EXAMPLES Example 1 FVIII Frameworks and Fusion Partners

The fusion proteins of the present invention consist of a FVIII protein (FVIII part) joined to a polypeptide (fusion partner) from another protein.

The FVIII part of the fusion protein can be any protein with FVIII activity. The FVIII part can be a B domain-deleted/truncated (BDD) FVIII protein, in which parts of the FVIII B domain has been removed from the protein. Non-limiting examples of FVIII frameworks that can constitute the FVIII part of fusion proteins is shown in Table 1. F8-500 is a BDD human FVIII protein. Starting at the N-terminus, F8-500 consists of FVIIIs signal peptide (amino acid −19 to −1) followed by FVIII HC without the B domain (amino acid 1-740), a 21 amino acid linker (SFSQNSRHPSQNPPVLKRHQR) (SEQ ID NO: 2), and FVIII LC (amino acid 1649-2332 of wild-type human FVIII. The sequence of the 21 amino acid linker is derived from the B domain of FVIII and consists of amino acid 741-750 and 1638-1648 of full-length wild-type human FVIII.

F8-500-Δa3 consists of F8-500 without the a3 region. In F8-500-┘a3 amino acid 1647-1687 of wild-type human FVIII is eliminated from F8-500. Thereby, the furin site at amino acid 1645-1648 is destroyed. A combined furin and thrombin site is, however, created by the R1645-H1646-P1688-R1689 amino acid stretch in F8-500-Δa. The a3 region is important for binding of FVIII to vWF and therefore, the affinity of F8-500-Δa3 for vWF is reduced compared to wild-type FVIII.

F8-500-His consists of F8-500 with a His tag inserted in the linker of F8-500. Thus the linker sequence of F8-500-His is SFSQNSRHPSHHHHHHSQNPPVLKRHQR (SEQ ID NO: 3).

F8-500-Δa3-His consists of F8-500 without the a3 region but with a His tag inserted in the linker of F8-500. Thus, in F8-500-Δa3-His amino acid 1647-1687 of wild-type human FVIII has been eliminated from F8-500 and the linker sequence is SFSQNSRHPSHHHHHHSQNPPVLKRHQR (SEQ ID NO: 4).

F8-500-Y1680F and F8-500-Y1680C consist of F8-500 in which amino acid 1680 of full-length wild-type human FVIII has been changed from tyrosine to phenylalanine and cysteine, respectively. Both these amino acid replacements reduce the affinity of FVIII to vWF factor. Furthermore, the Y1680C amino acid replacement introduces a free cysteine that can be used as a handle for conjugating protracting moieties to the fusion protein.

The fusion partner can be joined to several positions on the FVIII part of the fusion protein. Non-limiting examples of positions on FVIII for joining to the fusion partner are in the B domain or the B-domain-derived linker between the FVIII HC and LC, at the position of a3, and at the C-terminus of FVIII LC.

Example 2 Construction of Expression Vectors Encoding FVIII Frameworks and Fusion Proteins

The fusions between FVIII and fusions partners all involves PCR for amplifying the fusion partner. Restriction sites are added to the ends of the PCR primers used. Restriction enzymes are used for cloning of fusion partner cDNA or synthetic DNA into FVIII cDNA.

Fusions in the B-domain of F8-500 takes place between aa750 and aa1638. Restriction sites AvrII, NruI, AgeI and MluI within or flanking the B-domain are used for insertion of the fusion partner encoding DNA.

For fusions at the carboxy terminus of FVIII light chain, the F8-500 coding construct is modified. The internal BamHI site (aa 604-606) is eliminated by site-directed mutagenesis and DNA encoding the flexible (GGGS)₆ linker is inserted 3′ to the coding region. A new BamHI site is introduced in the 3′ end of the linker-coding DNA in order to ease cloning of C-terminal fusion partners between BamHI and NotI sites. Subsequently, fusion partner DNA is inserted. The fusions proteins derived from this construct is referred to as F8-500-C2-linked-(GGGS)6-X in Table 2-12 Similar to the (GGGS)6 linker a minimal GS-linker (BamHI restriction site) was inserted in the 3′ end of the F8-500 coding region. The BamHI restriction site (GGATCC) form the two codons of GS (Glycine-Serine). The fusions proteins derived from this construct is referred to as F8-500-C2-linked-GS-X in Table 2-12. Fusions to the C-terminus of F8-500 without any linker were made by PCR amplifying the fusions with extended primers harboring the last 109 bp of the F8-500 coding region in the 5′end and a NotI restriction site in the 3′end of the PCR product. The XbaI restriction site is present 104-109 bp from the F8-500 stop codon. XbaI and NotI restriction enzymes were used for cloning the fusion partners without linkers. The fusions proteins derived from these latter construct is referred to as F8-500-C2-linked-X in Table 2-12.

For insertion of the fusion partner coding DNA at a3 positions thus replacing a3 with the fusion partner in the encoded protein, the SacII restriction site is introduced 3′ to the coding region of a3. Thus, fusion partner coding DNA can be introduced by insertion between the AgeI and SacII sites or between the AvrII and SacII sites.

hFc (SEQ ID NO: 5) HTCPPCPAPEAEGEPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK mFc (SEQ ID NO: 6) KPCPPCKCPAPNAEGEPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVLTA QTQTHREDYQSTLRVVSALPIQHQDWMSGKEFKCKVNNKALPAPIERTISKPKGSVRAPQVYVL PPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEK KNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK Human Serum Albumin (HSA) (SEQ ID NO: 7) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCD KSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTA FHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGK ASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLEC ADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKN YAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLV EEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKR MPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTF HADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEG KKLVAASQAALGL Transferrin (SEQ ID NO: 8) VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEADAVTLDA GLVYDAYLAPNNLIKPVVAEFYGSKEDPQTFYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAG WNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAF KCLKDGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARS MGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYL GYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIA KIMNGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCEDTPEAGYFAVAVVKKSASDLTW DNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLN LCEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDG TRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLL FRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRP hFc_RI (CD64) (SEQ ID NO: 9) QVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDS GEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAF KFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVIKELFPAPVLNASVTSPLLEGNLVT LSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLIKRSP ELELQVLGLQLPTP FcRn (SEQ ID NO: 10) AESHLSLLYHLTAVSSPAPGTPAFWVSGWLGPQQYLSYNSLRGEAEPCGAWVWENQVSWYWE KETTDLRIKEKLFLEAFKALGGKGPYTLQGLLGCELGPDNTSVPTAKFALNGEEFMNFDLKQGT WGGDWPEALAISQRWQQQDKAANKELTFLLFSCPHRLREHLERGRGNLEWKEPPSMRLKARPS SPGFSVLTCSAFSFYPPELQLRFLRNGLAAGTGQGDFGPNSDGSFHASSSLTVKSGDEHHYCCIVQ HAGLAQPLRVELESPAKSS FcRn-H166K (SEQ ID NO: 11) AESHLSLLYHLTAVSSPAPGTPAFWVSGWLGPQQYLSYNSLRGEAEPCGAWVWENQVSWYWE KETTDLRIKEKLFLEAFKALGGKGPYTLQGLLGCELGPDNTSVPTAKFALNGEEFMNFDLKQGT WGGDWPEALAISQRWQQQDKAANKELTFLLFSCPHRLREKLERGRGNLEWKEPPSMRLKARPS SPGFSVLTCSAFSFYPPELQLRFLRNGLAAGTGQGDFGPNSDGSFHASSSLTVKSGDEHHYCCIVQ HAGLAQPLRVELESPAKSS LRP-CR5-6 (SEQ ID NO: 12) TCPPNQFSCASGRCIPISWTCDLDDDCGDRSDESASCAYPTCFPLTQFTCNNGRCININWRCDND NDCGDNSDEAGCSH LRP-CR6-7 (SEQ ID NO: 13) TCFPLTQFTCNNGRCININWRCDNDNDCGDNSDEAGCSHSCSSTQFKCNSGRCIPEHWTCDGDN DCGDYSDETHANCTNQATR LRP-CR6 (SEQ ID NO: 14) TCFPLTQFTCNNGRCININWRCDNDNDCGDNSDEAGCSH SC anti-GPIIIa-1-HC-LC (SEQ ID NO: 15) MDILMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKPGKSFMGLIYYGTNLVDGVPSRFSGS GSGADYSLTISSLDSEDFADYYCVQYAQLPYTFGGGTKLEKLGGGGSGGGGSGGGGSNSVQLQ QSGAELVKPGASVKLSCTASGFNIKDTYVHWVKQRPEQGLEWIGRIDPANGYTKYDPKFQGKA TITADTSSNTAYLQLSSLTSEDTAVYYCVRPLYDYYAMDYWGQGTSVTVSS Linker-SC anti-GPIIIa-1-HC-LC (SEQ ID NO: 16) MDILMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKPGKSFMGLIYYGTNLVDGVPSRFSGS GSGADYSLTISSLDSEDFADYYCVQYAQLPYTFGGGTKLEKLGGGGSGGGGSGGGGSNSVQLQ QSGAELVKPGASVKLSCTASGFNIKDTYVHWVKQRPEQGLEWIGRIDPANGYTKYDPKFQGKA TITADTSSNTAYLQLSSLTSEDTAVYYCVRPLYDYYAMDYWGQGTSVTVSSGGGGSGGGGSGG GGS SC anti-GPIIIa-2-HC-LC (SEQ ID NO: 17) QVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGDIYPGGGYNKYNEN FKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCAREYGNYDYAMDSWGQGTSVTVSSGGGGSG GGGSGGGGSDIVMTQAAPSVPVTPGESVSISCRSSRSLLHSNGNTYLCWFLQRPGQSPQLLIYRM SNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKR SC anti-GPIIIa-2-LC-HC (SEQ ID NO: 18) DIVMTQAAPSVPVTPGESVSISCRSSRSLLHSNGNTYLCWFLQRPGQSPQLLIYRMSNLASGVPDR FSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKRGGGGSGGGGSGGGGSQV QLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGDIYPGGGYNKYNENFK GKATLTADTSSSTAYMQLSSLTSEDSAVYFCAREYGNYDYAMDSWGQGTSVTVSS ABD035 (SEQ ID NO: 19) LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP Sequence A (SEQ ID NO: 20) GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG SAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTST EEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG SAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG ELP80 (SEQ ID NO: 21) GVPGVGVPGGGVPGVGVPGVGVPGAGVPGVGVPGGGVPGVGVPGVGVPGAGVPGVG VPGGGVPGVGVPGVGVPGAGVPGVGVPGGGVPGVGVPGVGVPGAGVPGVGVPGGGV PGVGVPGVGVPGAGVPGVGVPGGGVPGVGVPGVGVPGAGVPGVGVPGGGVPGVGVP GVGVPGAGVPGVGVPGGGVPGVGVPGVGVPGAGVPGVGVPGGGVPGVGVPGVGVPG AGVPGVGVPGGGVPGVGVPGVGVPGAGVPGVGVPGGGVPGVGVPGVGVPGAGVPGV GVPGGGVPGVGVPGVGVPGAGVPGVGVPGGGVPGVGVPGVGVPGAGVPGVGVPGGG VPGVGVPGVGVPGAGVPGVGVPGGGVPGVGVPGVGVPGAGVPGVGVPGGGVPGVGV PGVGVPGA Extra a3 (SEQ ID NO: 22) EITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPR GHBP (SEQ ID NO: 23) FSGSEATAAILSRAPWSLQSVNPGLKTNSSKEPKFTKCRSPERETFSCHWTDEVHHGTKNLGPIQL FYTRRNTQEWTQEWKECPDYVSAGENSCYFNSSFTSIWIPYCIKLTSNGGTVDEKCFSVDEIVQP DPPIALNWTLLNVSLTGIHADIQVRWEAPRNADIQKGWMVLEYELQYKEVNETKWKMMDPILT TSVPVYSLKVDKEYEVRVRSKQRNSGNYGEFSEVLYVTLPQMSQ FIX298-342 (SEQ ID NO: 24) IFLKFGSGYVSGWARVFHKGRSALVLQYLRVPLVDRATCLRSTKF FIX47-125 (SEQ ID NO: 25) DDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVV CSCTEGYRLAENQKSCE vWF1-272 (SEQ ID NO: 26) SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALER CPCFHQGKEYAPGETVKIGCNTCVCRDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGEC QYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVIKRPMKDETHF EVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDP VDFGNSWKVSSQCADTR vWF1-1390 (SEQ ID NO: 27) SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALER CPCFHQGKEYAPGETVKIGCNTCVCRDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGEC QYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVIKRPMKDETHF EVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDP VDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLD VCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRY NSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGK KVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHDFYCSRLL DLVFLLDGSSRLSEAEFEVLKAFVVDMMERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSEL RRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRIALLLMASQEPQRMSRNFVRYVQGLK KKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTLPPD MAQVTVGPGLLGVSTLGPKRNSMVLDVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSI HVTVLQYSYMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQ APNLVYMVTGNPASDEIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLV LQRCCSGEGLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQVSV LQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALGFAVRYLTSEMHGARPGASKAV VILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGPAGDSNVVKLQRIEDLPTM VTLGNSFLHKLCSGFVRICMDEDGNEKRPGDVWTLPDQCHTVTCQPDGQTLLKSHRVNCDRGL RPSCPNSQSPVKVEETCGCRWTCPCVCTGSSTRHIVTFDGQNFKLTGSCSYVLFQNKEQDLEVIL HNGACSPGARQGCMKSIEVKHSALSVELHSDMEVTVNGRLVSVPYVGGNMEVNVYGAIMHEV RFNHLGHIFTFTPQNNEFQLQLSPKTFASKTYGLCGICDENGANDFMLRDGTVTTDWKTLVQEW TVQRPGQTCQPILEEQCLVPDSSHCQVLLLPLFAECHKV vWF497-716-R545A (SEQ ID NO: 28) EDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDMMERLAISQKWVRVAVVEYHD GSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRIALLLMASQEP QRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVS YLCDLAPEAPPPTLPPDMAQVTVG vWF binding protein (SEQ ID NO: 29) NPELKDFNEEEQLKCDLELNKLENQILMLGKTFYQNYRDDVESLYSKLDLIMGYKDEERANKK AVNKRMLENKKEDLETIIDEFFSDIDKTRPNNIPVLEDEKQEEKNHKNMAQLKSDTEAAKSDESK RSKRSKRSLNTQNHKPASQEVSEQQKAEYDKRAEERKARFLDNQKIKKTPVVSLEYDFEHKQRI DNENDKKLVVSAPTKKPTSPTTYTETTTQVPMPTVERQTQQQIIYNAPKQLAGLNGESHDFITTH QSPTTSNHTHNN hCG C-terminus (SEQ ID NO: 30) SSSSKAPPPSLPSPSRLPGPSDTPILPQ F10AP (SEQ ID NO: 31) SVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQTQPERGDNNL

Example 3 Transient Expression of FVIII Frameworks and Fusion Proteins

HKB11 cells at a density of 0.9-1.1×10⁶ are transfected with a complex of plasmid 0.7 mg/l and the transfection agent, 293Fectin (Invitrogen) 1.4 ml/l. The transfection complex is prepared by diluting the plasmid and the transfection separately in OPTIMEM (Invitrogen), mixing of the two solutions, and incubation of the mixture at room temperature for 20 minutes. The complex mixture is added to the cell suspension and the suspension is incubated in shaker incubator for 5 days at 36.5° C. and 5% CO₂. The cell culture harvest is filtered on a 0.22 μm membrane filter. FVIII frameworks and fusion proteins are purified from the cell culture harvest as described in Example 5.

Example 4 Stable Cell Expressing Fusion Protein

Serum-free adapted CHO-DUKX-B11 cells were transfected with an expression plasmid encoding the F8-500-albumin-Δa3 fusion protein. Transfected cells were selected with the dihydrofolate reductase system and cloned by limiting dilution. Clones were screened for FVIII production by ELISA and chromogenic activity assay. The clone JsJH009 was seleceted for upscaling. The cells were transferred to a bioreactor. The F8-500-albumin-Δa3 fusion was purified from cell culture harvests as described in Example 5.

Example 5 Purification of FVIII Frameworks and Fusion Proteins

A column was packed with the resin VIIISelect (GE Healthcare), with the dimensions 1.6 cm in diameter and 4 cm in bed height giving 8 mL, and was equilibrated with 20 mM Imidazol+10 mM CaCl₂+0.01% Tween80+250 mM NaCl, pH7.3 at 500 cm/h. The culture filtrate prepared as described in Example 3 was applied to the column, and the column was subsequently washed with first equilibration buffer and then 20 mM Imidazol+10 mM CaCl₂+0.01% Tween80+1.5M NaCl, pH7.3. The bound FVIII was eluted isocratic at 90 cm/h with 20 mM Imidazol+10 mM CaCl₂+0.01% Tween80+2.5 M NaCl+6.5M Propylenglycol, pH7.3. The fractions containing FVIII were pooled and diluted 1:10 with 20 mM Imidazol+10 mM CaCl₂+0.01% Tween80, pH7.3 and applied to a column packed with F25-Sepharose (Thim et al., Haemophilia, 2009). The column dimension was 1.6 cm in diameter and 2 cm in bed height giving 4 mL in column volume. The column was equilibrated at 180 cm/h with 20 mM Imidazol+10 mM CaCl₂+0.01% Tween80+150 mM NaCl+1M Glycerol, pH7.3 prior to application. After application the column was washed first with equilibration buffer and then 20 mM Imidazol+10 mM CaCl₂+0.01% Tween80+650 mM NaCl, pH7.3. The bound FVIII was isocratic eluted with 20 mM Imidazol+10 mM CaCl₂+0.01% Tween80+2.5M NaCl+50% (v/v) Ethylenglycol, pH7.3 at 30 cm/h. The fractions containing FVIII were pooled and diluted 1:15 with 20 mM Imidazol+10 mM CaCl₂+0.01% Tween80, pH7.3, except FVIII-variants with deletions of the a3 domain which were diluted 1:45 in the same buffer. The diluted pool was applied to a column packed with Poros 50HQ (PerSeptive Biosystem), with the column dimensions 0.5 cm in diameter and 5 cm in bed height giving 1 mL in column volume. The column was equilibrated at 300 cm/h with 20 mM Imidazol+10 mM CaCl₂+0.01% Tween80+50 mM NaCl+1M Glycerol, pH7.3 prior to application. The column was washed with equilibration buffer before the elution using a linear gradient over 5 column volumes from equilibration buffer to 20 mM Imidazol+10 mM CaCl₂+0.01% Tween80+1M NaCl+1M Glycerol, pH7.3. The fractions containing FVIII were pooled and the pool was stored at −80° until use. A table of yields over a typical purification is shown in Table 11.

The FVIII-variants with HIS-tag was purified essentially as described above, however the second purification step (F25-sepharose) was exchanged to Chelating Sepharose FF (GE Healthcare) charged with 2 column volumes of 1M NiSO4. The column dimension was 0.5 cm in diameter and 5 cm bed height giving 1 mL column volume. The column was equilibrated with 30 mM Imidazol+10 mM CaCl₂+0.01% Tween80+1.5M NaCl, pH7.3 at 180 cm/h prior to application. After application the column was washed with 30 column volumes of equilibration buffer prior to elution using a linear gradient over 5 column volumes to 250 mM Imidazol+10 mM CaCl₂+0.01% Tween80+1.5M NaCl, pH7.3. The fractions containing FVIII were pooled and diluted 1:30 with 20 mM Imidazol+10 mM CaCl₂+0.01% Tween80, pH7.3. The final purification step (Poros 50HQ) was performed as described above.

Example 6 FVIII:C in Cell Culture Harvests Measured by Chromogenic Assay

The FVIII activity (FVIII:C) of the rFVIII compound in cell culture harvest (supernatant fraction) was evaluated in a chromogenic FVIII assay using Coatest SP reagents (Chromogenix) as follows: rFVIII samples and a FVIII standard (Coagulation reference, Technoclone) were diluted in Coatest assay buffer (50 mM Tris, 150 mM NaCl, 1% BSA, pH 7.3, with preservative). Fifty μl of samples, standards, and buffer negative control were added to 96-well microtiter plates (Spectraplates MB, Perkin Elmer). All samples were tested diluted 1:100, 1:400, 1:1600, and 1:6400. The factor IXa/factor X reagent, the phospholipid reagent and CaCl₂ from the Coatest SP kit were mixed 5:1:3 (vol:vol:vol) and 75 μl of this added to the wells. After 15 min incubation at room temperature, 50 μl of the factor Xa substrate 5-2765/thrombin inhibitor I-2581 mix was added and the reactions were incubated 5 min at room temperature before 25 μl 1 M citric acid, pH 3, was added. The absorbance at 405 nm was measured on an Envision microtiter plate reader (Perkin Elmer) with absorbance at 620 nm used as reference wavelength. The value for the negative control was subtracted from all samples and a calibration curve prepared by linear regression of the absorbance values plotted vs. FVIII concentration. The specific activity was calculated by dividing the activity of the samples with the protein concentration determined by ELISA. The results are shown in Table 1-10.

Example 7 Purification of FVIII Frameworks and Fusion Proteins

F8-500-albumin-Δa3 purified as described in Example 5 (13 mg, 6.5 mg/ml) in a buffer consisting of: imidazol (20 mM), calcium chloride (10 mM), Tween 80 (0.02%), sodium chloride (500 mM), and glycerol (1 M) in water (pH 7.3, was thawed.

Nine microgram sialidase from Arthrobacter ureafaciens (1.9 U, in 7 microliter buffer), 280 microgram sialyl tranferase (in 112 microliter, His-ST3Gal-I, 2.5 mg/ml, EC 2.4.99.4, WO 2006102652), and 59 milligram cytidine monophospate N-5′-PEG-glycerol-neuraminic acid (in 290 microliter, see WO2007/056191) were added, and pH was adjusted to 6.9 using HCl (1M). The final volume was 2.7 mL. The resulting mixture was then left for 22 hours at 22-25 degrees Celsius (room temperature).

After glycopegylation, the mixture was diluted to 50 ml with Buffer A (Tris (25 mM), calcium chloride (10 mM), Tween80 (0.02%), and glycerol (1 M) in water, pH 7.5). The deluted mixture was loaded onto a Source30Q column (GE Healthcare Bio-Sciences, Hillerød, Denmark, column volume 1.1 mL). The immobilised material was then washed with Buffer A (4 column volumes) and subsequently eluted from the column using a gradient of 0-100% Buffer B (Tris (25 mM), calcium chloride (10 mM), Tween 80 (0.02%), sodium chloride (0.7 M), and glycerol (1 M) in water, pH 7.5). Gradient: 8 CV 5-10% Buffer B, 13.5 CV 10-100% Buffer B, and 3 CV 100% Buffer B.

The early eluting 2.5 mL peak fraction was mixed with 1.2 milligram cytidine monophospate N-5′acetyl-neuraminic acid (in 12 microliter) and 62 microgram sialyltransferase (in 52 microliter, MBP-SBD-ST3Gal-III, EC 2.4.99.6, see WO 2006102652).

The mixture was left for 22 hours at 22-25 degrees Celsius (room temperature), and subsequently loaded onto a Superdex 200 pg column (GE Healthcare Bio-Sciences, Hillerød, Denmark; column volume 120 ml). Product was then eluted using a buffer consisting of: L-histidine (1.5 g/L), L-methionine (55 mg/L), calcium chloride (250 mg/L), Tween 80 (0.1 g/L), sodium chloride (18 g/L), and sucrose (1.5 g/L) in water, pH 6.9. The first product-containing fractions (12 mL) were isolated and pooled; product concentration was approximately 0.06 mg/mL.

Finally, the product was concentrated by centrifugation in an Amicon Centriprep YM-50 (cut-off: 50 kDa). The volume after concentration was 1.7 mL, containing 0.35 mg/mL glycopegylated F8-500-albumin-Δa3 (purity >90%). This compound is referred to in Table 12 as 40K-PEG-O-F8-500-albumin-Δa3.

Example 8 FVIII:C in Purified Samples Measured by Chromogenic Assay

The FVIII activity (FVIII:C) of the purified rFVIII compound (isolated as disclosed in Example 5) was evaluated in a chromogenic FVIII assay using Coatest SP reagents (Chromogenix) as follows: rFVIII samples and a FVIII standard (e.g. purified wild-type rFVIII calibrated against the 7th international FVIII standard from NIBSC) were diluted in Coatest assay buffer (50 mM Tris, 150 mM NaCl, 1% BSA, pH 7.3, with preservative). Fifty μl of samples, standards, and buffer negative control were added to 96-well microtiter plates (Nunc) in duplicates. The factor IXa/factor X reagent, the phospholipid reagent and CaCl₂ from the Coatest SP kit were mixed 5:1:3 (vol:vol:vol) and 75 μl of this added to the wells. After 15 min incubation at room temperature 50 μl of the factor Xa substrate S-2765/thrombin inhibitor 1-2581 mix was added and the reactions incubated 10 min at room temperature before 25 μl 1 M citric acid, pH 3, was added. The absorbance at 415 nm was measured on a Spectramax microtiter plate reader (Molecular Devices) with absorbance at 620 nm used as reference wavelength. The value for the negative control was subtracted from all samples and a calibration curve prepared by linear regression of the absorbance values plotted vs. FVIII concentration. The specific activity was calculated by dividing the activity of the samples with the protein concentration determined by HPLC. For HPLC, the concentration of the sample was determined by integrating the area under the peak in the chromatogram corresponding to the light chain and compare with the area of the same peak in a parallel analysis of a wild-type rFVIII, where the concentration was determined by amino acid analyses. The results are shown in Table 1-10.

Example 9 FVIII:C in Purified Samples Measured by One-Stage Clot Assay

FVIII:C of the rFVIII compounds was further evaluated in a one-stage FVIII clot assay as follows: rFVIII samples and a FVIII standard (e.g. purified wild-type rFVIII calibrated against the 7th international FVIII standard from NIBSC) were diluted in HBS/BSA buffer (20 mM hepes, 150 mM NaCl, pH 7.4 with 1% BSA) to approximately 10 U/ml followed by 10-fold dilution in FVIII-deficient plasma containing VWF (Dade Behring). The samples were subsequently diluted in HBS/BSA buffer. The APTT clot time was measured on an ACL300R or an ACL5000 instrument (Instrumentation Laboratory) using the single factor program. FVIII-deficient plasma with VWF (Dade Behring) was used as assay plasma and SynthASil, (HemosIL™, Instrumentation Laboratory) as aPTT reagent. In the clot instrument, the diluted sample or standard is mixed with FVIII-deficient plasma, aPTT reagents at 37° C. Calcium chloride is assed and time until clot formation is determined by turbidity. The FVIII:C in the sample is calculated based on a standard curve of the clot formation times of the dilutions of the FVIII standard. The results are shown in Table 1-10.

Example 10 Pharmacokinetics of FVIII Frameworks and Fusion Proteins in FVIII- and VWF-Deficient Mice

The pharmacokinetics of rFVIII variants were evaluated in FVIII-deficient mice (FVIII exon 16 knock out (KO) mice with C57Bl/6 background, bred at Taconic M&B) or in vWF-deficient mice (vWF exon 4+5 KO mice with C57Bl/6 background bred at Charles River, Germany) The vWF-KO mice had 13% of normal FVIII:C (see example 6), while the FVIII-KO mice had no detectable FVIII:C. A mixture of male and female (approximately 1:1) with an approximate weight of 25 grams and age range of 16-28 weeks were used. The mice received a single i.v. injections of rFVIII (280 IU/kg) in the tail vein. Blood was taken from the orbital plexus at time points up to 64 hours after dosing using non-coated capillary glass tubes. Three samples were taken from each mouse, and 2 to 4 samples were collected at each time point. Blood was immediately stabilized with sodium citrate and diluted in four volumes FVIII Coatest SP buffer (see example 6) before 5 min centrifugation at 4000×g. Plasma obtained from diluted blood was frozen on dry ice and kept at −80° C. The FVIII:C was determined in a chromogenic assay as described in example 6 Pharmacokinetic analysis was carried out by non-compartmental methods (NCA) using WinNonlin Pro version 4.1 software. Results are shown in Table 12. The fold prolongation of fusion protein is calculated by dividing the half-life of the fusion protein with that of the FVIII framework without the fusion partner.

Example 11 Analysis of Fusion Protein Binding to Platelets

Platelet-binding of a fusion protein can be tested by flow cytometry. Peripheral blood platelets may be purified, or whole blood can be used. The platelets may be activated or resting. The platelets are incubated with fusion protein for 15-30 min. The fusion protein may be directly labelled with a fluorophore or detected using a fluorescently labelled secondary antibody.

A fluorescently labelled platelet specific antibody not interfering with binding of the fusion protein can be added to assess whether the particles binding the fusion protein are indeed platelets. After incubation, the cells are washed to remove fusion protein, and the samples are analyzed on a flow cytometer. The flow cytometer detects unlabelled cells and fluorescently labelled molecules binding to cells and thus can be used to specifically analyze to which extent fusion protein is bound to platelets (or other cells).

The specificity of binding can be assessed e.g. by adding a surplus of unlabelled antibody (when using directly labelled fusion protein). Binding of the FVIII moiety to the platelets can be assessed e.g. by adding a surplus of annexin V or FVIII.

Internalization of the fusion protein by the resting platelet may be assessed e.g. by incubating platelets with directly labelled fusion protein followed by incubation with an antibody, which quenches the signal from surface-bound (i.e. not internalized) fusion protein. Only the internalized fusion protein will then be detected by flow cytometry. It may hypothesized that activated platelets will release internalized fusion-protein at the site of clot formation.

Example 12 Pharmacokinetics of GPIIIa-Targeted Fusion Proteins in GPIIIa Transgenic Mice

The GPIIIa-targeted fusion protein binds to the human GPIIb/IIIa (integrin a2b3) receptor on platelets, but it may not recognize murine GPIIb/IIIa, preventing the use of wild type mice for pharmacokinetic analyses. The pharmacokinetic profile of GPIIIa-targeted fusion proteins can be analyzed in transgenic mice expressing human GPIIIa, which associates with murine GPIIb enabling the binding of fusion protein to the receptor. Fusion protein will be injected intravenously to GPIIIa transgenic mice and blood collected at various time-points after injection (e.g. 0.5, 24, 72, 288 hours). The injected fusion protein (free and/or platelet-bound) may be quantified by means of an ELISA or the fusion protein may be radioactively or fluorescently labelled and detected.

TABLE 1 BDD FVIII frameworks that can constitute the FVIII part of fusion proteins Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500 F8-500 n.a n.a 17.9/17.0 F8-500-Δa3 F8-500-Δa3 n.a n.a 13.6/13.8 F8-500-His F8-500-His n.a. n.a. 12.1/8.1  F8-500-Δa3-His F8-500-Δa3- n.a. n.a. n.d./n.d. His F8-500-Y1680F F8-500- n.a. n.a. 16.6**/n.d   Y1680F F8-500-Y1680C F8-500- n.a. n.a. 19.7/n.d.  Y1680C *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE 2 Fusion proteins interacting with FcRn Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500-hFc(IgG1) F8-500 hFc In B domain 19.9**/n.d. 14.1**/n.d. F8-500-hFc(IgG1)-His F8-500-His hFc In B domain F8-500-Y1680F- F8-500- hFc In B domain hFc(IgG1) Y1680F F8-500-Y1680C- F8-500- hFc In B domain hFc(IgG1) Y1680C F8-500- F8-500-Δa3 hFc At a3s position  6.0**/n.d. hFc(IgG1)-Δa3 28.5**/n.d. F8-500-C2 linked- F8-500 hFC At C-terminus 16.7**/n.d. (GGGS)6-hFc(IgG1) of LC F8-500-C2 linked-GS- F8-500 hFC At C-terminus 14.4**/n.d. hFc(IgG1) of LC F8-500-C2 linked- F8-500 hFC At C-terminus 13.4**/n.d. hFc(IgG1) of LC (F8-500-hFC-LC) F8-500-Y1680F-C2- F8-500- hFC At C-terminus linked-hFc(IgG1) Y1680F of LC (F8-500-Y1680F-hFC- LC) F8-500-Y1680C-C2- F8-500- hFC At C-terminus linked-hFc(IgG1) Y1680C of LC (F8-500-Y1680C-hFC- LC) F8-500-mFc(IgG2A) F8-500 mFc In B domain 18.5**/n.d. 13.9**/n.d. F8-500-mFc(IgG2A)- F8-500-His mFc In B domain His F8-500-Y1680F- F8-500- mFc In B domain mFc(IgG2A) Y1680F F8-500-Y1680C- F8-500- mFc In B domain mFc(IgG2A) Y1680C F8-500- F8-500-Δa3 mFc At a3s position mFc(IgG2A)-Δa3 F8-500-C2 linked- F8-500 mFC At C-terminus 15.5**/n.d. (GGGS)6- of LC mFc(IgG2A) F8-500-C2 linked-GS- F8-500 mFC At C-terminus 15.2**/n.d. mFc(IgG2A) of LC F8-500-C2 linked- F8-500 mFC At C-terminus 12.9**/n.d  mFc(IgG2A) of LC F8-500-Y1680F-C2- F8-500- mFC At C-terminus linked-mFc(IgG2A) Y1680F of LC F8-500-Y1680C-C2- F8-500- mFC At C-terminus linked-mFc(IgG2A) Y1680C of LC F8-500-albumin F8-500 HSA In B domain  14.7/n.d. F8-500-albumin-His F8-500-His HSA In B domain   8.1/6.2 F8-500-Albumin- F8-500 HSA In B domain 12.3**/n.d. S727P F8-500-Y1680F- F8-500- HSA In B domain albumin Y1680F F8-500-Y1680C- F8-500- HSA In B domain albumin Y1680C F8-500-albumin-Δa3 F8-500-Δa3 HSA At a3s position   2.0/n.d. F8-500-C2 linked- F8-500 HSA At C-terminus 22.7**/n.d. (GGGS)6-albumin of LC F8-500-C2 linked-GS- F8-500 HSA At C-terminus 17.7**/n.d. albumin of LC F8-500-C2 linked- F8-500 HSA At C-terminus albumin of LC (F8-500-albumin-LC) F8-500-Y1680F-C2- F8-500- HSA At C-terminus linked-albumin Y1680F of LC (F8-500-Y1680F- albumin-LC) F8-500-Y1680C-C2- F8-500- HSA At C-terminus linked-albumin Y1680C of LC (F8-500-Y1680C- albumin-LC) F8-500-transferrin F8-500 Transferrin In B domain F8-500-transferrin-His F8-500-His Transferrin In B domain F8-500-Y1680F- F8-500- Transferrin In B domain transferrin Y1680F F8-500-Y1680C- F8-500- Transferrin In B domain transferrin Y1680C F8-500- F8-500-Δa3 Transferrin At a3s position transferrin-Δa3 F8-500-C2 linked- F8-500 Transferrin At C-terminus transferrin of LC (F8-500-transferrin- LC) F8-500-Y1680F-C2- F8-500- Transferrin At C-terminus linked- transferrin Y1680F of LC (F8-500-Y1680F- transferrin-LC) F8-500-Y1680C-C2- F8-500- Transferrin At C-terminus linked-transferrin Y1680C of LC (F8-500-Y1680C- transferrin-LC) *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE 3 Fusion proteins interacting with immunoglobulins Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500-hFcγRI F8-500 hFcγRI (CD64) In B domain   22.2/20.0 F8-500-hFcγRI-His F8-500-His hFcγRI (CD64) In B domain  11.6/4.9 F8-500-Y1680F- F8-500- hFcγRI (CD64) In B domain hFcγRI Y1680F F8-500-Y1680C- F8-500- hFcγRI (CD64) In B domain hFcγRI Y1680C F8-500- F8-500-Δa3 hFcγRI (CD64) At a3s position   5.3/n.d. hFcγRI-Δa3 F8-500-C2-linked- F8-500 hFcγRI (CD64) At C-terminus hFcγRI of LC (F8-500-hFcγRI-LC) F8-500-Y1680F-C2- F8-500- hFcγRI (CD64) At C-terminus linked-hFcγRI Y1680F of LC (F8-Y1680F-500- hFcγRI-LC) F8-500-Y1680C-C2- F8-500- hFcγRI (CD64) At C-terminus linked- hFcγRI Y1680C of LC (F8-500-Y1680C- hFcγRI-LC) F8-500-FcRn F8-500 FcRn In B domain 27.2**/n.d. F8-500-FcRn-H166K F8-500 FcRn In B domain 20.8**/n.d. *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE 4 Fusion proteins with the capability of reducing interaction with clearance receptors Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500-LRP-CR5-6 F8-500 LRP CR5-6 In B domain  14.2/n.d. F8-500-LRP-CR5-6- F8-500- LRP CR5-6 In B domain His His F8-500-LRP-CR6-7 F8-500 LRP CR6-7 In B domain 9.5**/n.d. F8-500-LRP-CR6 F8-500 LRP CR6 In B domain 8.9**/n.d. *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE 5 Fusion proteins binding to platelets Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500-anti-GPIIIa- F8-500 SC anti-GPIIIa-1- In B domain 29.3**/n.d. 1-HC-LC HC-LC F8-500-linker- anti- F8-500 SC anti-GPIIIa-1- In B domain 40.3**/n.d. GPIIIa-1-HC-LC HC-LC F8-500-SC anti- F8-500 SC anti-GPIIIa-2- In B domain 22.5**/n.d. GPIIIa-2-HC-LC HC-LC F8-500-SC anti- F8-500-His SC anti-GPIIIa-2- In B domain — GPIIIa-2-HC-LC-His HC-LC F8-500-linker-SC F8-500-His SC anti-GPIIIa-2- In B domain 45.9**/n.d. anti-GPIIIa-2-HC-LC- HC-LC His F8-500- 

 a3-SC anti- F8-500- SC anti-GPIIIa-2- In B domain — GPIIIa-2-HC-LC-His

 a3-His HC-LC F8-500-Y1680F-SC F8-500- SC anti-GPIIIa-2- In B domain anti-GPIIIa-2-HC-LC Y1680F HC-LC F8-500-Y1680C-SC F8-500- SC anti-GPIIIa-2- In B domain anti-GPIIIa-2-HC-LC Y1680C HC-LC F8-500-SC anti- F8-500- 

 a3 SC anti-GPIIIa-2- At a3s position 26.2**/n.d. GPIIIa-2-HC-LC-Δa3 HC-LC F8-500-C2-linked-SC F8-500 SC anti-GPIIIa-2- At C-terminus anti-GPIIIa-2-HC-LC HC-LC of LC (F8-500-SC anti- GPIIIa-2-HC-LC-LC) F8-500-Y1680F-C2- F8-500- SC anti-GPIIIa-2- At C-terminus linked-SC anti-GPIIIa- Y1680F HC-LC of LC 2-HC-LC (F8-500-Y1680F-SC anti-GPIIIa-2-HC-LC- LC) F8-500-Y1680C-C2- F8-500- SC anti-GPIIIa-2- At C-terminus linked-SC anti-GPIIIa- Y1680C HC-LC of LC 2-HC-LC (F8-500-Y1680C-SC anti-GPIIIa-2-HC-LC- LC) F8-500-SC anti- F8-500 SC anti-GPIIIa-2- In B domain 28.3**/n.d. GPIIIa-2-LC-HC LC-HC F8-500-SC anti- F8-500 SC anti-GPIIIa-2- In B domain 95.4**/n.d. GPIIIa-2-LC-HC LC-HC F8-500-linker-SC F8-500-His SC anti-GPIIIa-2- In B domain 47.5**/n.d. anti-GPIIIa-2-LC-HC- LC-HC His F8-500-Δa3-SC anti- F8-500- SC anti-GPIIIa-2- In B domain 81.4**/n.d. GPIIIa-2-LC-HC-His Δa3-His LC-HC F8-500-Y1680F-SC F8-500- SC anti-GPIIIa-2- In B domain anti-GPIIIa-2-LC-HC Y1680F LC-HC F8-500-Y1680C- SC F8-500- SC anti-GPIIIa-2 In B domain anti-GPIIIa-2-LC-HC Y1680C (LC-HC) F8-500-SC anti- F8-500-Δa3 SC anti-GPIIIa-2- At a3s position 29.2**/n.d. GPIIIa-2-LC-HC-Δa3 LC-HC F8-500-C2-linked- F8-500 SC anti-GPIIIa-2- At C-terminus 22.4**/n.d. (GGGS)6-SC anti- LC-HC of LC GPIIIa-2-LC-HC (F8-500-SC anti- GPIIIa-2-LC-HC-LC) F8-500-Y1680F-C2- F8-500- SC anti-GPIIIa-2- At C-terminus linked-SC anti-GPIIIa- Y1680F LC-HC of LC 2-LC-HC (F8-500-Y1680F-SC anti-GPIIIa-2-LC-HC- LC) F8-500-Y1680C-C2- F8-500- SC anti-GPIIIa-2- At C-terminus 16.6**/n.d. linked-GS-SC anti- Y1680C LC-HC of LC GPIIIa-2-LC-HC (F8-500-y1680C-SC anti-GPIIIa-2-LC-HC- LC) *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE 6 Fusion proteins interacting with serum albumin Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500-ABD035 F8-500 Albumin binding In B domain peptide ABD035 F8-500-ABD035-His F8-500-His Albumin binding In B domain  8.3/n.d. peptide ABD035 F8-500-Y1680F- F8-500- Albumin binding In B domain ABD035 Y1680F peptide ABD035 F8-500-Y1680C- F8-500- Albumin binding In B domain ABD035 Y1680C peptide ABD035 F8-500- F8-500- 

 a3 Albumin binding At a3s position 4.0/0.5 ABD035-Δa3 peptide ABD035 F8-500-C2-linked- F8-500 Albumin binding At C-terminus ABD035 peptide ABD035 of LC (F8-500-ABD035- LC) F8-500-Y1680F-C2- F8-500- Albumin binding At C-terminus linked ABD035 Y1680F peptide ABD035 of LC (F8-500-Y1680F- ABD035-LC) F8-500-Y1680C-C2- F8-500- Albumin binding At C-terminus linked ABD035 Y1680C peptide ABD035 of LC (F8-500-Y1680C- ABD035-LC) F8-500-4XABD035 F8-500 4 X Albumin In B domain 14.8/n.d.  binding peptide ABD035 F8-500-4XABD035- F8-500-His 4 X Albumin In B domain His binding peptide ABD035 F8-500-Y1680F-4X F8-500- 4 X Albumin In B domain ABD035 Y1680F binding peptide ABD035 F8-500-Y1680C-4X F8-500- 4 X Albumin In B domain ABD035 Y1680C binding peptide ABD035 F8-500- F8-500-Δa3 4 X Albumin At a3s position 4X ABD035-Δa3 binding peptide ABD035 F8-500-C2-linked-4X F8-500 4 X Albumin At C-terminus ABD035 binding peptide of LC (F8-500-4X ABD035 ABD035-LC) F8-500-Y1680F-C2- F8-500- 4 X Albumin At C-terminus linked-4X ABD035 Y1680F binding peptide of LC (F8-500-Y1680F-4X ABD035 ABD035-LC) F8-500-Y1680C-C2- F8-500- 4 X Albumin At C-terminus linked-4X ABD035 Y1680C binding peptide of LC (F8-500-Y1680C-4X ABD035 ABD035-LC) F8-500-SC anti-HSA F8-500 SC anti-HSA In B domain F8-500-SC anti-HSA- F8-500-His SC anti-HSA In B domain His F8-500-Y1680F-SC F8-500- SC anti-HSA In B domain anti-HSA Y1680F F8-500-Y1680C-SC F8-500- SC anti-HSA In B domain anti-HSA Y1680C F8-500- F8-500-Δa3 SC anti-HSA At a3s position SC anti-HSA-Δa3 F8-500-C2-linked-SC F8-500 SC anti-HSA At C-terminus anti-HSA of LC (F8-500-SC anti- HSA-LC) F8-500-Y1680F-C2- F8-500- SC anti-HSA At C-terminus linked-SC anti-HSA Y1680F of LC (F8-500-Y1680F-SC anti-HSA-LC) F8-500-Y1680C-C2- F8-500- SC anti-HSA At C-terminus linked-SC anti-HSA Y1680C of LC (F8-500-Y1680C-SC anti-HSA-LC) *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE 7 Fusion proteins shielding the molecule from clearance receptors Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500-Seq A F8-500 Sequence A In B domain F8-500-Seq A-His F8-500-His Sequence A In B domain 19.2**/n.d. F8-500-Y1680F-Seq F8-500- Sequence A In B domain A Y1680F F8-500-Y1680C-Seq F8-500- Sequence A In B domain A Y1680C F8-500- F8-500-Δa3 Sequence A At a3s position Seq A-Δa3 F8-500-C2-linked- F8-500 Sequence A At C-terminus Seq A of LC (F8-500-Seq A-LC) F8-500-Y1680F-C2- F8-500- Sequence A At C-terminus linked Seq A Y1680F of LC (F8-500-Y1680F-Seq A-LC) F8-500-Y1680C-C2- F8-500- Sequence A At C-terminus linked Seq A Y1680C of LC (F8-500-Y1680C-Seq A-LC) F8-500-ELP80 F8-500 ELP80 In B domain F8-500-ELP80-His F8-500-His ELP80 In B domain 13.8**/n.d. F8-500-Y1680F- F8-500- ELP80 In B domain ELP80 Y1680F F8-500-Y1680C- F8-500- ELP80 In B domain ELP80 Y1680C F8-500- F8-500-Δa3 ELP80 At a3s position ELP80-Δa3 F8-500-C2-linked- F8-500 ELP80 At C-terminus ELP80 of LC F8-500-Y1680F-C2- F8-500- ELP80 At C-terminus linked-ELP80 Y1680F of LC F8-500-Y1680C-C2- F8-500- ELP80 At C-terminus linked ELP80 Y1680C of LC *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE 8 Fusion proteins with modulated affinity to vWF Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500- Extra a3 F8-500 Extra a3 In B domain 10.0**/n.d. F8-500- Extra a3-His F8-500-His Extra a3 In B domain  7.7**/n.d. F8-500-Y1680F- F8-500- Extra a3 In B domain Extra a3 Y1680F F8-500-Y1680C- F8-500- Extra a3 In B domain Extra a3 Y1680C F8-500- F8-500-Δa3 Extra a3 At a3s position Extra a3-Δa3 F8-500-C2-linked- F8-500 Extra a3 At C-terminus Extra a3 of LC (F8-500-Extra a3-LC) F8-500-Y1680F-C2- F8-500- Extra a3 At C-terminus linked- Extra a3 Y1680F of LC (F8-500-Y1680F- Extra a3-LC) F8-500-Y1680C-C2- F8-500- Extra a3 At C-terminus linked Extra a3 Y1680C of LC (F8-500-Y1680C- Extra a3-LC) *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE 9 Other fusion proteins Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500-GHBP F8-500 Growth hormone In B domain 22.8**/n.d. binding protein F8-GHBP-His F8-500-His Growth hormone In B domain   7.6/4.5 binding protein F8-500-FIX298-342 F8-500 AS 298-342 of FIX In B domain 12.3**/n.d. F8-500-FIX47-125 F8-500 AS 47-125 of FIX In B domain  18.0/n.d. F8-500-FIX47-125- F8-500-His AS 47-125 of FIX In B domain   14.4/13.3 His F8-500-vWF1-272 F8-500 AS 1-272 of vWF In B domain 44.5**/n.d. F8-500-vWF1-1390 F8-500 AS 1-1390 of vWF In B domain 16.0**/n.d. F8-500-vWF497-716- F8-500 AS 497-716 of In B domain  11.5/8.8. R545A vWF F8-500-vWF497-716- F8-500-His AS 497-716 of In B domain 256.9**/n.d.  R545A-His vWF F8-500-vWF binding F8-500 vWF-binding In B domain 19.8**/n.d. protein protein F8-500-hCG C- F8-500-His 28 C-terminal AS In B domain  11.4/n.d. terminus-His of hCG b-chain F8-500- F8-500-Δa3 28 C-terminal AS At a3s position  18.2/n.d. hCG-C-terminus- of hCG b-chain Δa3 F8-500- F8-500 Activation peptide In B domain 22.5**/n.d. F10AP of hFX F8-500- F8-500-His Activation peptide In B domain  16.8/n.d. F10AP-His of hFX F8-500-Y1680F- F8-500- Activation peptide In B domain F10AP Y1680F of hFX F8-500-Y1680C- F8-500- Activation peptide In B domain F10AP Y1680C of hFX F8-500- F8-500-Δa3 Activation peptide At a3s position F10AP-Δa3 of hFX F8-500-C2-linked- F8-500 Activation peptide At C-terminus F10AP of hFX of LC (F8-500-F10AP-LC) F8-500-Y1680F-C2- F8-500- Activation peptide At C-terminus linked-F10AP Y1680F of hFX of LC (F8-500-Y1680F- F10AP-LC) F8-500-Y1680C-C2- F8-500- Activation peptide At C-terminus linked- F10AP Y1680F of hFX of LC (F8-500-Y1680C- F10AP-LC) *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE 10 Fusion proteins with modulated affinity to lipids Position of Specific Fusion fusion activity Name FVIII part partner partner (mU/ng)* F8-500- Extra C2 F8-500 Extra C2 In B domain F8-500- Extra C2-His F8-500-His Extra C2 In B domain . F8-500-Y1680F- F8-500- Extra C2 In B domain Extra C2 Y1680F F8-500-Y1680C- F8-500- Extra C2 In B domain Extra C2 Y1680C F8-500- F8-500-Δa3 Extra C2 At a3s position Extra C2-Δa3 F8-500-C2 linked- F8-500 Extra C2 At C-terminus 8.3**/n.d. (GGGS)6-extra C2 of LC F8-500-C2-linked- F8-500 Extra C2 At C-terminus 7.7**/n.d. GS-Extra C2 of LC F8-500-Y1680F-C2- F8-500- Extra C2 At C-terminus linked-Extra C2 Y1680F of LC F8-500-Y1680C-C2- F8-500- Extra C2 At C-terminus linked-Extra C2 Y1680C of LC *Chromogenic assay/clot assay **Measured in cell culture harvest (high values may represent poor detection by ELISA)

TABLE 11 Typical yields of the purification process described in Example 5 Yield/% Sample Vol./mL Activity/U*mL⁻¹ Step Total Culture filtrate 1650 17 100 100  Step1: Flow thru 1650 3.5 21 — Step1: Eluate pool 14 1021 51 51 Step2: Flow thru 133 19 17 — Step2: Eluate pool 8 820 45 23 Step3: Flow thru 117 0.9 2 — Step3: Eluate pool 1.5 3000 69 16

TABLE 12 In vivo half-lifes of FVIII frameworks and fusion proteins in FVIII- or vWF deficient mice Half-life Fold Half-life Fold in FVIII- prolongation in vWF- prolongation deficient in FVIII- deficient in vWF- Name mice (h)* deficient mice* mice (h)* deficient mice* F8-500 4.5/8.6 n.a. n.d./0.3  n.a. F8-500-Δa3 0.3/0.3 n.a. n.d. n.a. F8-500-His n.d. n.a. 0.1/0.1 n.a. F8-500-Δa3-His n.d. n.a. n.d. n.a. F8-500-Y1680F  0.5/n.d. n.a. n.d. n.a. F8-500-Y1680C n.d. n.a. n.d. n.a. F8-500-hFc(IgG1)-Δa3  0.7/n.d.  2.3/n.d. n.d. n.d F8-500-C2 linked-  9.9/n.d. 2.2./n.d  n.d n.d (GGGS)6-hFc(IgG1) F8-500-C2 linked- 15.0/n.d.  3.5/n.d n.d./1.1  n.d/4 (GGGS)6-mFc(IgG2A) F8-500-albumin 7.8/9.1 1.7/1.1 n.d. n.d. F8-500-albumin-His 6.7/6.0 ?/? 1.6/0.6  16/6 F8-500-albumin-Δa3  1.4/n.d.  4.7/n.d. n.d. n.d. 40K-PEG-O-F8-500- 12.0/n.d.   40/n.d. n.d. n.d. albumin-Δa3 F8-500-C2 linked- n.d/9.6 n.d./1.1  n.d. n.d (GGGS)6-albumin F8-500-C2 linked-GS-  8.0/n.d. 1.8./n.d  n.d./0.8  n.d/3 albumin F8-500-hFcγRI 9.2/9.7 2.0/1.1 n.d. n.d. F8-500-hFcγRI-His  10/n.d.  2.2/n.d. 1.7/1.2  17/12 F8-500-hFcγRI-Δa3  0.2/n.d.  0.7/n.d. n.d. n.d. F8-500-LRP-CR5-6 6.2/6.4 1.4/0.7 n.d. n.d F8-500-ABD035-His 10.3/—  ?/? n.d./0.8  n.d/8 F8-GHBP-His n.d n.d. 0.1/0.2  1/2 F8-500-FIX47-125-His 5.3/5.3 ?/? n.d. n.d. F8-500-F10AP-His n.d n.d. 1.0/0.8  10/8 *Chromogenic assay/ELISA 

1. A FVIII molecule fused to a fusion partner, wherein the fusion partner replaces the A3-domain of the Factor VIII molecule, is inserted into the B-domain of Factor VIII, or is inserted in the C-terminal end of the C2 domain in Factor FVIII.
 2. The FVIII molecule according to claim 1, wherein the fusion partner is albumin.
 3. The FVIII molecule according to claim 2, wherein said molecule is conjugated with a side group.
 4. The FVIII molecule according to claim 1, wherein said FVIII molecule is a B domain truncated variant comprising a B domain having the sequence as set forth in SEQ ID NO: 2, and wherein the fusion partner is an Fc domain that is inserted in the C-terminal end of the C2 domain in Factor FVIII.
 5. The FVIII molecule according to claim 4, wherein said Fc domain is a mutated Fc domain having reduced effector functions and/or increased affinity to the neonatal Fc receptor.
 6. The FVIII molecule according to claim 1, wherein the Factor VIII molecule is fused to an Fc receptor.
 7. The FVIII molecule according to claim 6, wherein said Fc receptor is FcγRI.
 8. The FVIII molecule according to claim 1, wherein the Factor VIII molecule has reduced vWF binding capacity.
 9. The FVIII molecule according to claim 1, wherein the FVIII fusion protein is conjugated with a side group.
 10. The FVIII molecule according to claim 9, wherein the side group is selected from one or more of the list group consisting of: hydrophilic polymers, peptides and hydrophobic side groups.
 11. A method of making a molecule according to claim 1, wherein said method comprises incubating a host cell encoding said molecule under appropriate conditions.
 12. A method for treatmenting of haemophilia comprising administering an effective amount of the molecule of claim 1 to a subject in need thereof.
 13. A pharmaceutical composition comprising a molecule according to claim
 1. 